The effect of the carbohydrate chains on the susceptibility of thyroxine-binding globulin (TBG) to denaturation was evaluated. Removal of 85% of the carbohydrate decreased the stability of the protein to guanidinium chloride or to acid. The initial structural change was irreversible, indicating that incorporation of carbohydrate during biosynthesis of TBG is not responsible for the metastability of the protein. Deglycosylated TBG was also analyzed by isoelectric focussing, which revealed the prsence of two protein bands as seen with desialaylated TBG. This microheterogeneity therefore appears to reside in the polypeptide chain.